JOURNAL OF CLINICAL MICROBIOLOGY, June 1977, P. 665-667 Copyright C 1977 American Society for Microbiology
Vol. 5, No. 6
Printed in U.S.A.
Cellular Fatty Acids of Peptococcus variabilis and Peptostreptococcus anaerobius C. WAYNE MOSS,* M. A. LAMBERT, AND G. L. LOMBARD Center for Disease Control, Atlanta, Georgia 30333
Received for publication 24 February 1977
Cellular fatty acids of Peptococcus variabilis and Peptostreptococcus anaerobius were identified by gas chromatography, mass spectrometry, and associated analytical techniques. Iso- and anteiso-branched-chain acids were major components in both species. A number of recent studies have shown that column was used primarily to separate isovarious microorganisms can be distinguished from anteiso-branched-chain acids (7). Fatty on the basis of their cellular fatty acids (3, 4, acid methyl ester peaks were tentatively identi10). Closely related species often can be easily fied by comparing retention times on each coldifferentiated by qualitative or large quantita- umn with retention times of methyl ester stantive differences in their cellular fatty acid con- dards (Applied Science Lab, State College, Pa.; tents (5, 8, 9). In preliminary studies of gram- Analabs, North Haven, Conn.; Supelco, Bellepositive anaerobic cocci, we noted that fatty fonte, Pa.). Final identification was established acid profiles ofPeptococcus variabilis and Pep- by a combination of techniques including hytostreptococcus anaerobius differed markedly drogenation (1), bromination (8), and mass from each other and from a variety of other spectrometry (12). Combined gas chromatograbacteria examined in this laboratory. Detailed phy-mass spectrometry of methyl esters was studies to identify the fatty acids of these two done with a DuPont instrument type 21-491B equipped with a combination electron impactorganisms have been completed. Lyophilized cultures of P. anaerobius (CDC- chemical ionization source. Isobutane was used 17642; Virginia Polytechnic Institute, VPI, as reagent gas for the chemical ionization 4329) and P. variabilis (CDC-16284; ATCC source. The chromatograms in Fig. 1 clearly show 14955) were reconstituted and plated onto Trypticase soy agar supplemented with defibrinated that there are major differences in the cellular sheep blood (5%), yeast extract (0.5%), hemin fatty acids of these organisms. Approximately (0.005%), and vitamin K1 (0.001%). Well-iso- 80% of the total fatty acids of P. anaerobius lated colonies were picked into thioglycolate were saturated branched-chain acids, and the broth (BBL, 0135C), and the cultures were remainder were saturated straight-chain acids. checked for purity by the procedures of Dowell This was confirmed by the fact that no change and Hawkins (2). Actively growing thioglyco- occurred in retention times of any peak in the late cultures were inoculated into 200 ml of chromatogram upon acetylation, hydrogenaSchaedler broth (BBL), which was used for pro- tion, or bromination, indicating the absence of ducing cells for fatty acid analysis. After incu- hydroxy, unsaturated, and cyclopropane acids bation in an anaerobic atmosphere (85% N2, (1, 8). The branched methyl group position in 10% H2, 5% C02) for 24 to 36 h at 35°C, cells the fatty acid chain was first indicated by analwere removed by centrifugation and washed ysis on the ethylene glycol adipate column, where iso-branched-chain esters are eluted three times with distilled water. After centrifugation, the cells were saponi- slightly before their anteiso-homologue (7). Refied and the fatty acids were methylated by the tention time data from this column indicated procedure described previously (8). Methyl es- that the major peaks in P. anaerobius were isoters were analyzed on a Perkin-Elmer model branched-chain acids 10:0, 12:0, 14:0, and 16:0; 900 gas chromatograph (Perkin-Elmer, Nor- anteiso-branched-chain acids 11:0, 13:0, and walk, Conn.) equipped with a flame ionization 15:0 were also present. The identity of each of detector and a disc integrator recorder. Sam- these acids was confirmed by comparing its ples were analyzed on a nonpolar 3% OV-1 mass spectrum with authentic standards. Elecmethyl silicone column and on a polar 15% tron impact mass spectra of anteiso-branched ethylene glycol adipate column as described methyl esters were clearly distinguished from previously (7, 8). The ethylene glycol adipate iso-branched and normal methyl esters by com665
666
J. CLIN. MICROBIOL.
NOTES
i-10: :0
i-12:0
1 2:CD
Peptostreptococcus aneerobius
8:0 i-16-0 i-14:0
14:0
LU
z
0 U) ULJ
16:0
a-13:0
10:0
a-
28
26
24
22
aXl:
15:0
20
18
16
14
12
10
8
6
4
2
0
20
18
16
14 MIN
12
10
8
6
4
2
0
Peptococcus variabilis
18:1 1800
20:1
26
t
24
22
FIG. 1. Gas chromatogram of esterified fatty acids from saponified whole cells of P. anaerobius (top) and P. variabilis (bottom). Analysis was made on a 0.16-inch (4.06-mm ID) by 12-ft (3.66 m) coiled glass column packed with 3% OV-1 methyl silicone. Helium was used as carrier gas at a flow rate of 50 ml/min. The column temperature was 160°C, and, after injection of the sample, it was programmed to 265°C at a rate of 5°C/min. Sensitivity of the flame ionization detector was 2.5 x 10-12 A; attenuation, 32 X 102; peak designation numbers: before colon refers to the number of carbon atoms, to the right refers to the number of double bonds; Br, branched-chain acids; i-, a methyl group at the penultimate carbon atom; and a-, a methyl group at the antepenultimate carbon.
paring the ratio of me = M-29 and me = M-31 peaks in the electron impact spectra. With anteiso-esters, the M-29 is equal to or greater than the M-31, whereas the M-31 peak is approximately two times the size of the M-29 peak in iso-branched- and normal straight-chain esters (12, 13). Acids less than 15 carbons in length were absent, or present in only trace amounts, in P.
variabilis (Fig. 1). Monounsaturated 18:1 and 20:1 acids present in this organism were confirmed by hydrogenation and mass spectrometry. Relatively large amounts of branchedchain acids (15:0, 17:0, 19:0) were present. Gasliquid chromatographic analysis on the ethylene glycol adipate column (7) showed both isoand anteiso-isomers for each branched-chain acid in a ratio of approximately 2:1 (iso-an-
VOL. 5, 1977
NOTES
teiso). Identities of these acids were confirmed by comparing the me = M-29 and mie = M-31 relationship by mass spectrometry (12, 13). Although branched-chain fatty acids have been found in other bacteria (6, 11), the presence of large amounts of iso-branched-chain acids 10:0, 12:0, and 14:0, as compared with amounts of other acids present, appears unique to P. anaerobius. However, additional strains of this and other species of anaerobic cocci must be studied to establish the usefulness of cellular fatty acids as a means of distinguishing this group of organisms.
4.
5.
6. 7.
ADDENDUM A report of the cellular fatty acids of various species of anaerobic cocci was recently published (C.L. Wells and C.R. Field, J. Clin. Microbiol. 4: 515-521, 1976). Although many of the fatty acids were not identified and different growth media were used, the gas chromatographic profiles for P. anaerobius and P. variabilis were comparable to those shown in Fig. 1. LITERATURE CITED 1. Brian, B. L., and E. W. Gardner. 1968. Fatty acids from Vibrio cholerae lipids. J. Infect. Dis. 118:47-53. 2. Dowell, V. R., Jr., and T. M. Hawkins. 1974. Laboratory methods in anaerobic bacteriology. In CDC laboratory manual, publication no. (CDC) 74-8272. U.S. Government Printing Office, Washington, D.C. 3. Jantzen, E. T., T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. VI. Fatty acid composition of strains within
8.
9.
10.
11. 12.
13.
667
micrococcaceae. Acta Pathol. Microbiol. Scand. Sect. B 82:785-798. Jantzen, E., K. Bryn, T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. V. Fatty acid composition of Neisseriae and Moraxellae. Acta Pathol. Microbiol. Scand., Sect. B 82:767-779. Kaltenbach, C. M., C. W. Moss, and R. E. Weaver. 1975. Cultural and biochemical characteristics and fatty acid composition of Pseudomonas diminuta and Pseudomonas vesiculare. J. Clin. Microbiol. 1:339344. Kaneda, T. 1967. Fatty acids in the genus Bacillus. I. Iso- and anteiso-fatty acids as characteristic constituents oflipids in 10 species. J. Bacteriol. 93:894-903. Moss, C. W., and W. B. Cherry. 1968. Characterization of the C,, branched-chain fatty acids of Corynebacterium acnes by gas chromatography. J. Bacteriol. 95:241-242. Moss, C. W., and S. B. Dees. 1975. Identification of microorganisms by gas chromatographic-mass spectrometric analyses of cellular fatty acids. J. Chromatogr. 112:595-604. Moss, C. W., V. R. Dowell, Jr., V. J. Lewis, and M. A. Schekter. 1967. Cultural characteristics and fatty acid composition of Corynebacterium acnes. J. Bacteriol. 94:1300-1305. Prefontaine, G., and F. L. Jackson. 1972. Cellular fatty acid profiles as an aid to the classification of "corroding bacilli" and certain other bacteria. Int. J. Syst. Bacteriol. 22:210-217. Raines, L. J., C. W. Moss, D. Farshtchi, and B. Pittman. 1968. Fatty acids of Listeria monocytogenes. J. Bacteriol. 96:2175-2177. Ryhage, R., and E. Stenhagen. 1960. Mass spectrometry in lipid research. J. Lipid Res. 1:361-390. Tyrrell, D. 1968. The fatty acid composition of some Entomophthoraceae. H. The occurrence of branchedchain fatty acids in Conidiobolus denaesporus Drechsl. Lipids 3:368-372.
Vol. 5, No. 6
Printed in U.S.A.
Cellular Fatty Acids of Peptococcus variabilis and Peptostreptococcus anaerobius C. WAYNE MOSS,* M. A. LAMBERT, AND G. L. LOMBARD Center for Disease Control, Atlanta, Georgia 30333
Received for publication 24 February 1977
Cellular fatty acids of Peptococcus variabilis and Peptostreptococcus anaerobius were identified by gas chromatography, mass spectrometry, and associated analytical techniques. Iso- and anteiso-branched-chain acids were major components in both species. A number of recent studies have shown that column was used primarily to separate isovarious microorganisms can be distinguished from anteiso-branched-chain acids (7). Fatty on the basis of their cellular fatty acids (3, 4, acid methyl ester peaks were tentatively identi10). Closely related species often can be easily fied by comparing retention times on each coldifferentiated by qualitative or large quantita- umn with retention times of methyl ester stantive differences in their cellular fatty acid con- dards (Applied Science Lab, State College, Pa.; tents (5, 8, 9). In preliminary studies of gram- Analabs, North Haven, Conn.; Supelco, Bellepositive anaerobic cocci, we noted that fatty fonte, Pa.). Final identification was established acid profiles ofPeptococcus variabilis and Pep- by a combination of techniques including hytostreptococcus anaerobius differed markedly drogenation (1), bromination (8), and mass from each other and from a variety of other spectrometry (12). Combined gas chromatograbacteria examined in this laboratory. Detailed phy-mass spectrometry of methyl esters was studies to identify the fatty acids of these two done with a DuPont instrument type 21-491B equipped with a combination electron impactorganisms have been completed. Lyophilized cultures of P. anaerobius (CDC- chemical ionization source. Isobutane was used 17642; Virginia Polytechnic Institute, VPI, as reagent gas for the chemical ionization 4329) and P. variabilis (CDC-16284; ATCC source. The chromatograms in Fig. 1 clearly show 14955) were reconstituted and plated onto Trypticase soy agar supplemented with defibrinated that there are major differences in the cellular sheep blood (5%), yeast extract (0.5%), hemin fatty acids of these organisms. Approximately (0.005%), and vitamin K1 (0.001%). Well-iso- 80% of the total fatty acids of P. anaerobius lated colonies were picked into thioglycolate were saturated branched-chain acids, and the broth (BBL, 0135C), and the cultures were remainder were saturated straight-chain acids. checked for purity by the procedures of Dowell This was confirmed by the fact that no change and Hawkins (2). Actively growing thioglyco- occurred in retention times of any peak in the late cultures were inoculated into 200 ml of chromatogram upon acetylation, hydrogenaSchaedler broth (BBL), which was used for pro- tion, or bromination, indicating the absence of ducing cells for fatty acid analysis. After incu- hydroxy, unsaturated, and cyclopropane acids bation in an anaerobic atmosphere (85% N2, (1, 8). The branched methyl group position in 10% H2, 5% C02) for 24 to 36 h at 35°C, cells the fatty acid chain was first indicated by analwere removed by centrifugation and washed ysis on the ethylene glycol adipate column, where iso-branched-chain esters are eluted three times with distilled water. After centrifugation, the cells were saponi- slightly before their anteiso-homologue (7). Refied and the fatty acids were methylated by the tention time data from this column indicated procedure described previously (8). Methyl es- that the major peaks in P. anaerobius were isoters were analyzed on a Perkin-Elmer model branched-chain acids 10:0, 12:0, 14:0, and 16:0; 900 gas chromatograph (Perkin-Elmer, Nor- anteiso-branched-chain acids 11:0, 13:0, and walk, Conn.) equipped with a flame ionization 15:0 were also present. The identity of each of detector and a disc integrator recorder. Sam- these acids was confirmed by comparing its ples were analyzed on a nonpolar 3% OV-1 mass spectrum with authentic standards. Elecmethyl silicone column and on a polar 15% tron impact mass spectra of anteiso-branched ethylene glycol adipate column as described methyl esters were clearly distinguished from previously (7, 8). The ethylene glycol adipate iso-branched and normal methyl esters by com665
666
J. CLIN. MICROBIOL.
NOTES
i-10: :0
i-12:0
1 2:CD
Peptostreptococcus aneerobius
8:0 i-16-0 i-14:0
14:0
LU
z
0 U) ULJ
16:0
a-13:0
10:0
a-
28
26
24
22
aXl:
15:0
20
18
16
14
12
10
8
6
4
2
0
20
18
16
14 MIN
12
10
8
6
4
2
0
Peptococcus variabilis
18:1 1800
20:1
26
t
24
22
FIG. 1. Gas chromatogram of esterified fatty acids from saponified whole cells of P. anaerobius (top) and P. variabilis (bottom). Analysis was made on a 0.16-inch (4.06-mm ID) by 12-ft (3.66 m) coiled glass column packed with 3% OV-1 methyl silicone. Helium was used as carrier gas at a flow rate of 50 ml/min. The column temperature was 160°C, and, after injection of the sample, it was programmed to 265°C at a rate of 5°C/min. Sensitivity of the flame ionization detector was 2.5 x 10-12 A; attenuation, 32 X 102; peak designation numbers: before colon refers to the number of carbon atoms, to the right refers to the number of double bonds; Br, branched-chain acids; i-, a methyl group at the penultimate carbon atom; and a-, a methyl group at the antepenultimate carbon.
paring the ratio of me = M-29 and me = M-31 peaks in the electron impact spectra. With anteiso-esters, the M-29 is equal to or greater than the M-31, whereas the M-31 peak is approximately two times the size of the M-29 peak in iso-branched- and normal straight-chain esters (12, 13). Acids less than 15 carbons in length were absent, or present in only trace amounts, in P.
variabilis (Fig. 1). Monounsaturated 18:1 and 20:1 acids present in this organism were confirmed by hydrogenation and mass spectrometry. Relatively large amounts of branchedchain acids (15:0, 17:0, 19:0) were present. Gasliquid chromatographic analysis on the ethylene glycol adipate column (7) showed both isoand anteiso-isomers for each branched-chain acid in a ratio of approximately 2:1 (iso-an-
VOL. 5, 1977
NOTES
teiso). Identities of these acids were confirmed by comparing the me = M-29 and mie = M-31 relationship by mass spectrometry (12, 13). Although branched-chain fatty acids have been found in other bacteria (6, 11), the presence of large amounts of iso-branched-chain acids 10:0, 12:0, and 14:0, as compared with amounts of other acids present, appears unique to P. anaerobius. However, additional strains of this and other species of anaerobic cocci must be studied to establish the usefulness of cellular fatty acids as a means of distinguishing this group of organisms.
4.
5.
6. 7.
ADDENDUM A report of the cellular fatty acids of various species of anaerobic cocci was recently published (C.L. Wells and C.R. Field, J. Clin. Microbiol. 4: 515-521, 1976). Although many of the fatty acids were not identified and different growth media were used, the gas chromatographic profiles for P. anaerobius and P. variabilis were comparable to those shown in Fig. 1. LITERATURE CITED 1. Brian, B. L., and E. W. Gardner. 1968. Fatty acids from Vibrio cholerae lipids. J. Infect. Dis. 118:47-53. 2. Dowell, V. R., Jr., and T. M. Hawkins. 1974. Laboratory methods in anaerobic bacteriology. In CDC laboratory manual, publication no. (CDC) 74-8272. U.S. Government Printing Office, Washington, D.C. 3. Jantzen, E. T., T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. VI. Fatty acid composition of strains within
8.
9.
10.
11. 12.
13.
667
micrococcaceae. Acta Pathol. Microbiol. Scand. Sect. B 82:785-798. Jantzen, E., K. Bryn, T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. V. Fatty acid composition of Neisseriae and Moraxellae. Acta Pathol. Microbiol. Scand., Sect. B 82:767-779. Kaltenbach, C. M., C. W. Moss, and R. E. Weaver. 1975. Cultural and biochemical characteristics and fatty acid composition of Pseudomonas diminuta and Pseudomonas vesiculare. J. Clin. Microbiol. 1:339344. Kaneda, T. 1967. Fatty acids in the genus Bacillus. I. Iso- and anteiso-fatty acids as characteristic constituents oflipids in 10 species. J. Bacteriol. 93:894-903. Moss, C. W., and W. B. Cherry. 1968. Characterization of the C,, branched-chain fatty acids of Corynebacterium acnes by gas chromatography. J. Bacteriol. 95:241-242. Moss, C. W., and S. B. Dees. 1975. Identification of microorganisms by gas chromatographic-mass spectrometric analyses of cellular fatty acids. J. Chromatogr. 112:595-604. Moss, C. W., V. R. Dowell, Jr., V. J. Lewis, and M. A. Schekter. 1967. Cultural characteristics and fatty acid composition of Corynebacterium acnes. J. Bacteriol. 94:1300-1305. Prefontaine, G., and F. L. Jackson. 1972. Cellular fatty acid profiles as an aid to the classification of "corroding bacilli" and certain other bacteria. Int. J. Syst. Bacteriol. 22:210-217. Raines, L. J., C. W. Moss, D. Farshtchi, and B. Pittman. 1968. Fatty acids of Listeria monocytogenes. J. Bacteriol. 96:2175-2177. Ryhage, R., and E. Stenhagen. 1960. Mass spectrometry in lipid research. J. Lipid Res. 1:361-390. Tyrrell, D. 1968. The fatty acid composition of some Entomophthoraceae. H. The occurrence of branchedchain fatty acids in Conidiobolus denaesporus Drechsl. Lipids 3:368-372.
